The 2009 influenza (flu) pandemic caused by the A(H1N1)pdm09 virus and the sporadic human cases of highly pathogenic avian flu A virus infection emphasize the need for improved flu vaccines for both seasonal and potential pandemic flu strains. One approach to flu vaccination entails the nasal administration of live attenuated flu viruses (LAIV), which mimics, in part, the natural route and site of viral infectionin the human nasal epithelial cells (HNEC) of the upper respiratory tract. However, LAIV fail to induce a robust systemic immune response compared to natural wild-type (wt) flu infections, suggesting important differences in the host innate and adaptive immune responses to attenuated and wt viruses. Flu virus replication in HNEC results in the induction of host immune responses within both epithelial and leukocyte cell subsets of the nasal mucosa. On the other hand, flu viruses encode multiple strategies to regulate and suppress the initial host innate response in both a strain- and cell type-specific manner, which is an important determinant of flu pathogenicity. We hypothesize that measurement of (a) early host transcriptional responses to LAIV vs. wt strains in HNEC as well as in mucosal and peripheral lymphocyte populations and (b) peripheral plasmablast B cell and serum and nasal antibody responses from the same individuals will identify specific early immune markers predictive of viral replication, immunogenicity, and ultimately protection. We propose to take advantage of an ongoing flu A(H1N1)pdm09 challenge study at the NIH Clinical Center to test this hypothesis. We will collect two additional nasal swabs from study subjects and sort nasal epithelial and lymphocyte cell populations by flow cytometry for subsequent high-throughput microfluidic qRT-PCR analysis of mucosal immune responses and local viral replication. Blood and nasal wash samples are also being collected from the same individuals to measure peripheral transcriptional responses and systemic B cell and antibody responses. The specific aims are to: 1) Characterize early host immune transcriptional responses in bulk unsorted nasal mucosal cells and sorted HNEC and nasal lymphocyte populations and in peripheral blood cells, to identify common and unique transcriptional signatures induced by wt virus and compare them to the host responses to LAIV immunization, which is being studied with similar approaches in other ongoing studies at Stanford; and 2) Characterize peripheral plasmablast B cell and serum and mucosal antibody responses to wt flu infection at a quantitative level in these same individuals and compare these to other ongoing studies of LAIV at Stanford, to identify local and/or systemic early transcriptional innate immunity response markers that correlate with subsequent plasmablast and other adaptive local and systemic humoral immunity indicators. These studies are expected to generate new knowledge on the underlying mechanism for protective immunity against flu viruses, which will guide the development of improved seasonal and pandemic flu vaccines.